Fc500

VK2/E6E7 and DC2.4 cells were placed into a VWR 35-mm confocal dish (2 × 10⁵ cells each). After overnight culture to allow cells to attach to the dish, the medium was replaced with 10% CVM containing Cy5-labeled gene complexes (300 ng DNA per dish) for 1, 3, and 6 h. The nucleus was stained with Hoechst 3334 15 min before the observations. Subsequently, the cells were washed twice with cold PBS to remove complexes that were not taken. The intracellular localization of gene complexes was observed using CLSM (Cy5 excitation, 633 nm; emission, 670 nm).
Cellular uptake was further verified quantitatively using a flow cytometer (FC500, BD Biosciences). The VK2/E6E7 and DC2.4 cells were added into a 12-well plate (1 × 10⁵ cells per well). After the aforementioned treatment, the cells were collected at 1, 3, and 6 h. After thorough washing, the cells were resuspended in 0.5 mL PBS for analysis.

Cell cycle progression was analyzed using PI. To harvest the 3D spheroid colonies in the soft fibrin gels, the gels were washed three times with PBS and treated with Dispase^® II for 20 min at 37°C.19 (link) The cells were centrifuged at 1500 rpm for 5 min and fixed in cold 70% ethanol for 24 h at 4°C. The following day, the fixed cells were centrifuged at 1500 rpm for 10 min, and the pellet was resuspended in 1 mL PBS, centrifuged, and resuspended in 1 mL PI staining solution and RNase A. After incubation at 37°C for 15−30 min in the dark, the cells were analyzed by FACS (FC500, BD Biosciences, CA) to determine the cell cycle stage.9 (link) A concentration of 10 μg/mL camptothecin was used as a positive control.